Small molecule inhibitors of botulinum neurotoxins

ABSTRACT

The invention provides potent quinolinol-based BoNT/A small-molecule inhibitors of botulinum neurotoxins, in particular of  Clostridium botulinum  serotype A neurotoxins. The invention also provides methods of using these small-molecule inhibitors to inhibit infections by  Clostridium botulinum , as well as, methods of preventing infections by  Clostridium botulinum  through materials that may be ingested.

RELATED APPLICATIONS

This application relates to and claims priority to U.S. ProvisionalPatent Application No. 61/056,664, which was filed May 28, 2008, iscommonly owned, and is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a sequence listing in paper format and incomputer readable format, the teachings and content of which areincorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods and compositions for inhibitingbotulinum neurotoxins. Specifically, the invention providesquinolinol-based compositions and methods of using quinolinol-basedcompositions to inhibit the toxic effects of serotype A Clostridiumbotulinum neurotoxins.

Description of the Related Art

Botulinum neurotoxins (BoNTs), produced by the anaerobic, Gram-positivebacteria Clostridium botulinum, C. baratii, and C. butyricum, consist ofseven immunologically distinct serotypes (types A-G). Botulinumneurotoxins are synthesized as ˜150-kDa single-chain protoxins that arepost-translationally processed by proteolytic cleavage to form adisulfide-linked dimer composed of a 100-kDa heavy chain (HC) and a50-kDa light chain (LC) (27, 31, 35, 36). The HC comprises a 50-kDaC-terminal domain (Hc) that participates in the binding of toxin toproductive ectoacceptors on the cell surface of peripheral cholinergicnerve cells (3). Toxin is taken up into the cell by receptor-mediatedendocytosis (4) and the 50-kDa N-terminal domain (Hn) of the HCfacilitates the translocation of the LC across an endosomal membraneinto the cytosol of the nerve cell (30). The LC is a zinc-dependentendopeptidase that cleaves and inactivates SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) proteins:SNAP-25, VAMP/synaptobrevin, and syntaxin (31, 36). SNARE proteins areessential for exocytosis of neurotransmitter, and cleavage of SNAREprotein(s) by BoNT inhibits the release of acetylcholine from synapticterminals leading to neuromuscular paralysis or botulism (31, 35).

Worldwide, about 1000 cases of human BoNT poisoning, predominantlycaused by serotypes A and B, are reported yearly (17). In spite ofadvances in food production and storage/handling processes, cases offood-borne botulism persist, including a massive outbreak in Thailand(26) and the recent US botulism scare associated with canned chili andother products (2, 28).

Therapy for botulism consists of immunological intervention toneutralize and clear toxin from the circulation, and supportive care,which may include intubation and ventilatory assistance. However, whileantibody therapy can be very effective, it has several limitations,including limited availability, lot-to-lot potency variability and shortwindow of application. Clearly, there is a need for improved therapiesand compounds.

Since small-molecules can be potentially used to treat pre- andpost-exposure BoNT intoxication, research efforts to identify theseantagonists have dramatically increased in recent years. However, thediscovery and development of BoNT serotype A (BoNT/A) small-moleculeinhibitors have long challenged researchers. Part of the difficulty inthis endeavor can be attributed to the unusually large peptidesubstrate-enzyme interface (8) that requires a small-molecule with highaffinity to effectively block substrate binding (47). Moreover, the BoNTtoxin and its domains show considerable conformational flexibility,making design of effective inhibitors complicated. Despite thesechallenges, a number of papers have been published on the initial stepsto discover and develop inhibitors of BoNT/A protease activity usingdifferent approaches. Using high throughput screening of the NCIDiversity Set, as well as a series of 4-aminoquinolines, Burnett et al.(11) identified several small-molecule inhibitors of BoNT/A, from whicha common pharmacophore was predicted using molecular modeling (9).Similarly, a high throughput screen of a library of hydroxamates (6)resulted in the selection of 4-dichlorocinnamic hydroxamate as a leadstructure for further development (5). Capkova et al. (12) structurallymodified 2, 4-dichlorocinnamic acid hydroxamate to improve its potency.On the other hand, a computational screen of 2.5 million compoundsresulted in the identification of an inhibitor with a K_(i) of 12 μM(32), but this value was later invalidated (47). Computer-aidedoptimization of this inhibitor resulted in an analog that showed atwo-fold improvement in inhibitory potency and displayed competitivekinetics by chelating the active site zinc atom (47).

Though the above approaches have resulted in the identification of anumber of small-molecule BoNT/A inhibitors, no compound has yet advancedto pre-clinical development. The majority of these leads have only beendemonstrated to be effective in enzymatic assays (11, 12, 29, 32, 47).Only a few small-molecules have been tested in cell-based assays (5, 9,15) that involved mixing the compound with the toxin, and not bypre-loading the inhibitor. To date, none of the recently-identifiedBoNT/A inhibitors has been tested in a tissue-based system, and to dateonly two compounds were reported to have minimal in vivo activity (15).

Herein, are provided the identification of potent quinolinol-basedBoNT/A small-molecule inhibitors using an integrated strategy thatcombined in silico screening and successive biochemical tests, includingenzymatic (HPLC-based), cell-based, and tissue-based assays.

SUMMARY OF THE INVENTION

The invention provides compositions for inhibiting botulinumneurotoxins, preferably a neurotoxin associated with Clostridiumbotulinum serotype A. The invention also provides methods of using thesecompositions to inhibit botulinum neurotoxins and of treating a subjectexposed to botulinum neurotoxins.

Compositions of the invention comprise Formula 1, pharmaceuticallyacceptable salts of Formula 1, or combinations thereof. Formula 1 is

wherein R₁, R₂, and R₃, being the same or different, are each selectedfrom the group consisting of a H, a C₁ to C₆ straight or branched alkylgroup, an aryl group, a halogen, NR₅R₆, NO₂, CH₂OH, CHO, COOH, CN, SO₃H,and SO₂NR₇R₈; wherein R₅, R₆, R₇, and R₈, being the same or different,are each selected from the group consisting of a H, a C₁ to C₆ straightor branched alkyl group, and an aryl group; wherein R₄ is selected fromthe group consisting of a straight or branched alkyl group and an arylgroup; wherein Art is selected from the group consisting of a 5- and6-membered aromatic and heteroaromatic rings and polycyclic aromatic andheteroaromatic ring systems; wherein X is selected from the groupconsisting of a O, OCH₂, S, SCH₂, NR₉, NR₉CH₂, NR₉CO, and CH₂; andwherein R₉ is selected from the group consisting of a H, a C₁ to C₆straight or branched alkyl group, and an aryl group.

Claimed compositions of the invention exclude known compounds, forexample the known compounds listed in Tables 1 and 2 herein are notclaimed as compositions of the invention. Previously unknown analogs ofthe compounds listed in Tables 1 and 2 that inhibit botulinumneurotoxins are within the scope of the invention. Exemplary analogs areprovided in Table 3. Preferably the compositions of the invention havelittle or no toxicity to a subject exposed to the compositions.

Suitable halogens for inclusion in the invention, alone or incombination, are chloro (Cl), fluoro (F), bromo (Br), and iodo (I).

Preferred aryl groups for inclusion in the invention, alone or incombination, are phenyl, pyridyl, thiophenyl, furyl, or indolyl rings.

An aspect of the invention is that compositions of the invention mayinclude a pharmaceutically acceptable carrier, excipient, diluent, oradjuvant.

The invention also provides methods of using compositions comprisingFormula 1, pharmaceutically acceptable salts of Formula I, orcombinations thereof. Specifically, the invention provides methods ofinhibiting a botulinum neurotoxin in a subject by administering to thesubject a composition comprising at least one compound, orpharmaceutically acceptable salt, of Formula 1. Compositions of theinvention may be administered alone or as part of a therapy to treatbotulism, i.e. an infection by C. botulinum.

In another aspect of the invention, the methods of the invention may beused to inhibit or prevent a C. botulinum infection, in particular a C.botulinum serotype A infection, by admixing one or more compositions ofFormula 1, or a pharmaceutically acceptable salt thereof, with asubstance suspected of including or being exposed to C. botulinum priorto such substance being ingested by a subject or such substancecontacting another substance that is expected to be ingested by asubject.

In another aspect, compositions of the invention comprise Formula 2,pharmaceutically acceptable salts of Formula 2, or combinations thereof.Formula 2 is

wherein X is O, S, Se, or NH; Y is CH or N; and Ar₁ is a monocyclic orbicyclic aromatic or heteroaromatic ring system, or a biphenyl orbipyridyl ring system, or a bridged biphenyl or bipyridyl ring system,any of which may be further substituted by one or more halogens (F, Cl,Br, I), NR₁R₂, OR₃, SO₂NR₄R₅, SR₆, R₇; and wherein R₁, R₂, R₃, R₄, R, R₆and R₇, being the same or different, are each selected from the groupconsisting of H, a C₁ to C₆ straight or branched chain or cycloalkylring, a C₁ to C₆ straight or branched chain acyl group, or an arylgroup.

In a further aspect methods of the invention include using compositionscomprising Formula 2, pharmaceutically acceptable salts of Formula 2, orcombinations thereof. Specifically, the invention provides methods ofinhibiting a botulinum neurotoxin in a subject by administering to thesubject a composition comprising at least one compound, orpharmaceutically acceptable salt, of Formula 2. Compositions of theinvention may be administered alone or as part of a therapy to treatbotulism, i.e. an infection by C. botulinum.

In another aspect of the invention, the methods of the invention may beused to inhibit or prevent a C. botulinum infection, in particular a C.botulinum serotype A infection, by admixing one or more compositions ofFormula 2, or a pharmaceutically acceptable salt thereof, with asubstance suspected of including or being exposed to C. botulinum priorto such substance being ingested by a subject or such substancecontacting another substance that is expected to be ingested by asubject.

The invention also provides kits suitable for use with methods of theinvention. Such kits include at least one composition of Formula 1,Formula 2, a pharmaceutically acceptable salt of either Formula 1 orFormula 2, or a combination thereof. Kits of the invention may alsoinclude instructions, a container for a composition of the invention,and components such as an adjuvant, diluent, pharmaceutically acceptablecarrier and the like that may be admixed with a composition of theinvention.

“Alkyl” alone or in combination means from 1 to 15, preferably 1 to 6,carbon atoms. Unless otherwise specified, an alkyl group is inclusive ofa straight chain alkyl, branched alkyl, or cycloalkyl.

“Acyl” denotes groups —C(O)R, where R is hydrogen, lower alkyl,substituted lower alkyl, aryl, substituted aryl and the like.

“Aryl” refers to any functional group or substituent derived from asimple aromatic ring, may it be phenyl, pyridyl, furyl, thiopheneyl,indolyl, quinolinyl, etc. Simple aryl groups include phenyl, C₆Hs, whichis derived from benzene, the tolyl group, CH₃C₆H₄, which is derived fromtoluene (methylbenzene), and the pyridyl group, C₅H₄N, which is derivedfrom pyridine. Preferred aryl groups are simple aryl groups.

Herein, a “subject” is an animal or human. “Animal” refers to a fish,bird, or mammal, preferably the animal is a mammal such as a cat, dog,ungulate (e.g. horse, zebra, donkey, cattle/bison, rhinoceros, camel,hippopotamus, goat, swine, sheep, giraffe, okapi, moose, deer, tapir,antelope, or gazelle), rodent (e.g. mice, rats, and other small, gnawingmammals), bat, bear, primate, or cetacean.

Herein, “to inhibit or prevent a C. botulinum infection” means that onemore symptoms associated with a C. botulinum infection is reduced by atleast 10% in a subject as compared to a subject that receives notherapeutic treatment for a C. botulinum infection.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs at the time of filing. All patentsand publications referred to herein are incorporated by referenceherein.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The application contains at leastone drawing executed in color. Copies of this patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1. Integrated experimental flow-chart to identify BoNT/Asmall-molecule inhibitors.

FIG. 2 shows the efficacy of small-molecules in inhibiting SNAP-25cleavage.

FIG. 2A. The reaction products were analyzed on SDS-PAGE gels. Lane 1,S25 alone; Lanes 2-7 contained S25+rELC; Lane 3, +NSC 84096; Lane 4, +CB7967495; Lane 5, +CB 7969312; Lane 6, +NSC 84094; Lane 7, +CB 79698218.FIGS. 2B and 2C. Inhibition of BoNT/A-mediated SNAP-25 cleavage bysmall-molecules in cell-based assay. Efficacy of five small-moleculeinhibitors at 15 mM (FIG. 2B) and at 10 mM (FIG. 2C) is shown: Lane 1(With BoNT/A), Lane 2 (Without BoNT/A), Lanes 3-7 contained BoNT/A withinhibitor; Lane 3 (+NSC 84096), Lane 4 (+CB 7967495), Lane 5 (+CB7969312), Lane 6 (+NSC 84094), Lane 7 (+CB 7968218). These blotsrepresent three independent experiments.

FIG. 3. Effects of BoNT/A small-molecule inhibitors in mouse phrenicnerve hemidiaphragm assay (MPNHDA). In all assays, BoNT/A (+) and NoToxin (Δ) controls were run to demonstrate the difference in twitchtension between BoNT-intoxicated and normal tissues (n=18 for bothgroups). The twitch tension time courses are the averages of triplicateassays. The time courses include only the effective concentration ofinhibitor required for maximum protection, except for CRATKML which wasnot effective at the tested concentration: (▪) CB 7969312 (500 nM), (▴)CB 7967495 (5 μM), (∘) NSC 84094 (10 μM), (♦) CB 7968218 (10 μM), (x)NSC 84096 (20 μM), and (●) CRATKML (20 M).

FIG. 4. Binding mode of NSC 84094 into BoNT/ALC substrate binding cleftshowing the quinolinol group (light green) interacting with the Zn atom(light blue), while the pyridyl substituent can form hydrogen bond withArg363. Atom colors: nitrogen (blue), oxygen (red), hydrogen (white),enzyme C atoms (dark green) and inhibitor C atoms (light green).

DETAILED DESCRIPTION

The invention provides small-molecule inhibitors of C. botulinumneurotoxin serotype A (BoNT/A), as well as, methods of using these smallmolecule inhibitors to inhibit or treat botulism.

Eight small-molecule inhibitors of BoNT serotype A were initiallyidentified by using in silico screening of the NCI database followed byHPLC protease assays (see Table 1). A substructure/similarity search wasused to identify analogs of the lead hit NSC 1010 (see Table 2), andfocus was placed on five of its most potent analogs, i.e., NSC 84094,NSC 84096, CB 7967495, CB 7968218 and CB 7969312 (see Table 3). Not onlywere these five analogs highly effective against the full-length BoNT/ALC in the HPLC-based assays, they were also very active against atruncated form (1-425) of BoNT/A LC. The extent of inhibition and theIC₅₀ values of these leads were comparable to or even better than thoseof previously reported small-molecule inhibitors (5, 6, 9-12, 15, 29,32). No small-molecule is known to have been previously found to beactive against both full-length and truncated forms of BoNT/A LC.Currently, it still remains unclear what form of light chain exists inthe cytosol during the actual BoNT catalysis of SNARE proteins.

The crystal structures of the endopeptidase of different serotypes ofBoNT are very similar. The HEXXH (SEQ ID NO: 4) motif that ischaracteristic of the catalytic site of Zn-endopeptidase is conserved.For instance, the active sites of BoNT/A and BoNT/B endopeptidasesdiffer only in two residues: F162 and F193 in BoNT/A correspond to N169and S200 in BoNT/B (32). Thus, it is conceivable for some BoNT/Ainhibitors also to be active against other serotypes. Here, the fiveanalogs exhibited cross reactivity, albeit reduced, against BoNT/B LC.Such a finding is not unprecedented. The two inhibitors described byTang et al. (47) were also reported to inhibit BoNT/B LC atconcentrations >20 μM. Interestingly, all five compounds (at 240 μM)failed to inhibit BoNT serotype E light chain. This finding seems tosuggest that they are more selective to serotype A.

It is also significant that efficacy of these candidate inhibitors wasobserved against BoNT/A holotoxin in cellular assays. Cells treated withall five compounds showed protection from the deleterious effects BoNT/Ahad on SNAP-25 at concentrations lower than the effective levels ofpreviously reported small-molecule inhibitors (5, 9, 15). Of furtherimportance is the observation that these five compounds exhibited littleor no cell toxicity, a characteristic that is highly desirable in thedevelopment of a therapeutic drug. Unlike several BoNT/A small-moleculeinhibitors that were reported cytotoxic at >5 μM (15) or >40 μM (9),four of the compounds (NSC 84096, CB 7969312, NSC 84094, and CB 7967495)were well-tolerated by the cells at concentrations up to 50 μM, and one(CB 7968218) exhibited signs of toxicity (aggregation and detachment ofN2a cells from the plate surfaces) only at concentrations above 45 μM.

The findings in the tissue-based assay revealed that all five compoundswere highly effective in the MPNHDA, significantly delaying theBoNT/A-induced paralytic half-time by at least threefold, while thepeptide BoNT/A inhibitor Ac-CRATKML-NH₂ (SEQ ID NO: 1) was notprotective. Among the five lead inhibitors, CB 7969312 was the mosteffective, at 0.5 μM. This striking observation represents CB 7969312 tobe the most potent small-molecule BoNT/A inhibitor reported to date thatexhibited activity in a tissue-based assay. While the in vivo mousebioassay is preferred for evaluating the efficacy of candidate BoNTinhibitors, during the early phase of drug discovery and development,the ex vivo MPNHDA has a potential over the in vivo assay in that itrequires only two mice per sample tested, and the results can beavailable within hours.

The effectiveness of these inhibitors in the in vitro and ex vivo assayswas only demonstrated when the compound was premixed with BoNT/A toxin;pre-loading the inhibitor did not protect cells/tissues against BoNTintoxication. To date, no one is known to have been able to provideexperimental evidence showing that inhibitors work in a pre-loadingsystem. The small-molecule inhibitor that was reported to be active inprimary neurons (9) was demonstrated to show a dose-dependent inhibitionof SNAP-25 cleavage in a non-pre-loading system (cells were pretreatedwith inhibitor for 45 minutes followed by incubation with BoNT/A in thecontinuous presence of inhibitor). Additionally, the inhibitors reportedby Eubanks et al. (15) and Boldt et al. (7) were characterized in cellculture assays that involved mixing BoNT/A toxin and varyingconcentrations of inhibitor.

The small-molecule inhibitors identified herein have three aromaticgroups, one of which is an 8-quinolinol moiety. Quinolinol is known tochelate divalent cations (14) and also has antimicrobial properties (18,19). Because of potential indiscriminate metal chelation, the8-hydroxyquinoline motif has been tagged as a “not suitable” functionalcomponent of BoNT/A LC inhibitors (1). However, the particular class ofquinolinol identified herein (with the other two ring systems and asecondary amine, see Tables 2 and 3) displayed specificity for BoNTserotype A and did not inhibit simply by chelating active-site zinc.

The structures of the quinolinol derivatives also contain additionalbasic moieties, including 2-amino or 3-amino pyridine (NSC 84094, CB7967495, CB 7968218, and CB 7969312). The presence of these structuralmotifs suggests that these lead inhibitors may interact with thehydrophobic pocket located in the active site of the LC (see FIG. 4).Adler et al. (1) reported that the quinolinol moiety alone in thepresence of zinc did not inhibit the proteolytic activity of BoNTserotypes A and B.

While the mechanism by which these newly identified small-moleculesinhibit BoNT/A is still being elucidated, molecular docking provided keyinsights into the likely binding sites and mode of inhibition. As shownin FIG. 4, NSC 84094 is docked in the large hydrophobic pocket of theBoNT/A LC active site, and its hydroxyquinoline moiety coordinates withzinc, which could explain the importance of this group in inhibitingBoNT/A LC, and suggests that the quinolinols inhibit BoNT/A by blockingthe active site zinc. Additionally, the pyridyl ring can form a hydrogenbond with Arg363, and may contribute to the specificity and potency ofthe inhibitor. It should be noted that the crystal structures of thecomplexes of known small-molecule and peptide inhibitors with BoNT/A LChave shown that chelation to zinc is involved in the binding andinhibition of the light chain in both cases (45, 46). A small moleculeinhibitor reported to have some in vivo efficacy (15) is a zinc chelator(5). Chelation appears to be a necessary but not sole condition of knowninhibitors. Further studies are warranted to fully determine the precisemechanism of action of these compounds.

In summary, the five small-molecule compounds represent highly potent,non-toxic BoNT/A inhibitors that were identified using an integratedscreening strategy. These compounds effectively inhibited the proteaseactivities of both BoNT/A LC (full-length and truncated), and alsosignificantly neutralized BoNT/A holotoxin in N2a cells andhemidiaphragm assays. Such protection at the cellular and tissue levelsis particularly important, since previously reported potent BoNT/A LCinhibitors such as Ac-CRATKML-NH₂ (SEQ ID NO: 1) were ineffective underthese conditions. Moreover, the effective inhibition by these compoundsof BoNT/A LC, but not BoNT/E LC, as well as their reduced efficiency ofBoNT/B LC, suggest that these small-molecules preferentially interactwith BoNT/A LC.

Pharmaceutical Preparations

Examples of a pharmaceutically acceptable carrier that may be used forpreparation of the compositions of the present invention include variousorganic or inorganic carriers that are conventionally used as apharmaceutical material. For example, excipients, lubricants, bindersand disintegrators can be used for solid preparations; and solvents,solubilizing agents, suspending agents, isotonic agents, buffer agents,soothing agents, etc., can be used for liquid preparations. Ifnecessary, conventional additives such as preservatives, antioxidants,coloring agents, sweetening agents, adsorbing agents and wetting agentscan be also used in an appropriate amount. Those of skill in the artwill be familiar with the carriers, diluents, adjuvants, etc. that arerecommended for the subject to whom compositions of the invention are tobe administered.

Administration to a Subject

Similarly, those of skill in the art will recognize that the preferreddosage to be administered to a subject will depend upon the species,gender, age, weight, and other health aspects of the subject. Inaddition, if the compositions of the invention are administered as partof a combination therapy for botulism or infection by a C. botulinum,the effects of the other therapeutic substances, alone and incombination, must be considered in determining the proper dosage andtime of administration to the subject.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and Methods

Initial test compounds were obtained from the Drug Synthesis andChemistry Branch, Developmental Therapeutics Program, Division of CancerTreatment and Diagnosis, National Cancer Institute (NCI; Bethesda, Md.),Sigma-Aldrich (St. Louis, Mo.) and Chembridge (CB) Corporation (SanDiego, Calif.). Compounds that passed the preliminary HPLC screeningwere synthesized and purified by GLSynthesis, Inc. (Worcester, Mass.).The chemical structure and purity (>98%) of these analogs were verifiedand confirmed by LC-MS and NMR prior to use in subsequent assays.Molecular weights of the compounds were confirmed by mass spectrometry.All compounds tested were racemic mixtures.

The BoNT/A peptide inhibitor (Ac-CRATKML-NH₂) (SEQ ID NO: 1) waspurchased from EMD Chemicals, Inc. (La Jolla, Calif.). Recombinantfull-length BoNT/A and BoNT/B LCs were prepared according to procedurespreviously described by Gilsdorf et al. (20) and Jensen et al. (24),both of which are incorporated by reference, and were >97% pure based onSDS-PAGE gels. The recombinant light chain for the type E neurotoxin(rELC; residues 1-423, SEQ ID NO: 8) and truncated type A light chain(tALC, residues 1-425, SEQ ID NO: 6) were cloned, expressed, andpurified. Briefly, rELC is encoded by the nucelotide sequence providedin SEQ ID NO: 7; tALC is encoded by the nucelotide sequence provided inSEQ ID NO: 5. rELC with a C-terminal 6×His-tag, and tALC were cloned andexpressed in E. coli (pET24a+/BL21(DE3)). Purification of rELC was byaffinity chromatography, followed by anion exchange chromatography.Purification of tALC involved a three-step ion exchange chromatographyusing Poros HS, Poros HQ, and Source 15S columns. The purity of rELC andtALC exceeded 90% and 97%, respectively, as judged by SDS/PAGE. Proteinconcentration was measured by BCA using BSA as a standard.

Botulinum neurotoxin serotype A (Hall strain) was obtained fromMetabiologics (Madison, Wis.). The specific toxicity of the toxin was2.4×10⁸ mouse i.p. LD₅₀/mg of protein, as determined by a toxintitration procedure described previously (25). Synthetic peptides usedas substrates for the HPLC assays were custom synthesized to >98% purityby Quality Controlled Biochemicals (Hopkinton, Mass.). The Alliance HPLCSystem (2695 XE Separation Module and 2996 Photodiode Array Detector)and the Empower/Millenium software were from Waters (Milford, Mass.).HPLC columns (Hi-Pore C18, 0.45×25 cm) were obtained from Bio-RadLaboratories (Hercules, Calif.). Anti-SNAP-25 mouse monoclonal IgG₁(SMI-81) was from CRP, Inc. (Berkeley, Calif.) and goat anti-mousehorseradish-peroxidase-conjugated was from KPL, Inc. (Gaithersburg,Md.). Cell culture media and reagents were from Lonza (Walkersville,Md.). ECL Advance Western Blotting Detection Kit was from GE Healthcare(Piscataway, N.J.). Tyrode's buffer was purchased from Sigma (St. Louis,Mo.).

Virtual Screening of BoNT/a Inhibitors

The three-dimensional structure of BoNT/A LC (PDB code: 1E1H) (39)obtained from the Protein Databank was used for virtual screening sinceit was the only suiTable 1 vailable crystal structure at the time the insilico screening was performed. One of the protomers was removed tovacate the active site. All water molecules were removed. Hydrogens andall-atom Kollman charges were added using the BIOPOLYMER module fromSYBYL. The three-dimensional structures of small-molecules for dockingwere generated using Concord as implemented in SYBYL. DOCK 4.0 (16) wasused for docking. Zinc parameters were optimized using the same trainingset as described by Hu et al. (23). In all cases, the scoring grids weredefined to include the whole active site around the Zn. Ligand fittingwith DOCK was employed using anchor-first docking with matching receptorsites, and using 25 peripheral seeds, 500 orientations, and uniformsampling. Anchors were first minimized, followed by layer 2 and thewhole ligand. Ten thousand minimization steps were done for furtherrefinement. All-atom representation and Gasteiger-Marsili empiricalatomic partial charges were used for the ligands.

HPLC-Based BoNT/a and BoNT/B LC Protease Assays

Selected compounds from virtual screening were tested in HPLC-basedBoNT/A LC and BoNT/B LC enzymatic assays as described previously by Gulet al. (21) and Schmidt and Bostian (37), both of which are incorporatedherein by reference. The BoNT/A LC assay mixture contained 50 mM HEPES(pH 7.3), 0.8 mM substrate containing residues 187-203 of SNAP-25(Ac-SNKTRIDEANQRATKML-NH₂) (SEQ ID NO: 2) (37), test compound dissolvedin dimethylsulfoxide (DMSO) at 10× the final assay concentration, and4.5-6.0 μg/mL (110-140 nM) BoNT/A LC. The LC catalyzes the hydrolysis ofSEQ ID NO: 2 between residues Q11 and R12 corresponding to residues Q11and R12 corresponding to residues 197 and 198 of SNAP-25.

The BoNT/B LC reaction mixture contained 50 mM HEPES buffer (pH 7.3),0.4 mM substrate corresponding to residues 60-94 of human VAMP-2(Ac-LSELDDRADALQAGASQFETSAAKLKRKYWWKNLK-NH₂) (SEQ ID NO: 3) (44), 1 mMDTT, test compound in DMSO and 1.5-2 μg/mL (30-40 nM) BoNT/B LC. The LCcatalyzes the hydrolysis of SEQ ID NO: 3 between residues Q17 and F18,corresponding to residue 76 and 77 of human VAMP-2. In control assays,the test compound was replaced by DMSO. The reaction was immediatelymixed upon adding the light chain, and incubated at 37° C. for 5 min.Assays were stopped by acidification with 90 μl of 0.7% trifluoroaceticacid (TFA). The amount of uncleaved substrate and products were thenmeasured after separation by reverse-phase HPLC. Solvent A was 0.1% TFAand solvent B was 70% acetonitrile/0.1% TFA. For assays with SEQ ID NO:2, the flow rate was 1.0 mL/min. at 25° C., with a gradient profile of10% B (2.5 min.), linear gradient to 36% B (21 min.), and 100% B (6min.). For assays with SEQ ID NO: 3 the flow rate was 1.0 mL/min. at 25°C., with a gradient profile of 20% B (2.5 min.), linear gradient to 80%B (21 min.), and 100% B (6 min.).

HPLC-Based BoNT/A and/B Holotoxin Assay

The BoNT/A toxin assay was based on the method developed by Schmidt andBostian (37) with some modification. Specifically, bovine serum albumin(BSA) was only used as a component of the buffer to dilute the toxin,hence its final concentration in the assay was reduced from 1 mg/ml to167 μg/ml. Sukonpan et al. (50) reported that BSA binds to a number oforganic compounds and could mask the identification of potentialinhibitors. Briefly, the assay mixture contained 30 mM HEPES buffer (pH7.3), 0.5 mM dithiothreitol, 0.25 mM ZnCl₂, 167 μg/ml of BSA, 0.8-0.9 mMsubstrate, 200 μM test compound in DMSO, and 335 nM of BoNT/A toxin.Immediately upon adding the holotoxin, each reaction mixture was mixedand incubated at 37° C. for 30 min. the reaction was stopped byacidification with trifluoroacetic acid (TFA). The amount of uncleavedsubstrate and products were then measured after separation byreverse-phase HPLC.

The BoNT/B toxin assay is similar to the BLC assay described above withthe following modifications: a) the BLC was replaced by BoNT/B toxindiluted in 0 mM HEPES, pH 7.2+10 mM DTT+20 μM ZnCl₂; final B toxinconcentration in the assay was 5.5. nM, and b) the reaction mixture wasmixed and incubated at 37° C. for 30 minutes.

Determination of the IC₅₀

The 50% inhibitory concentration (IC₅₀) values against BoNT/A LC werecalculated from nine concentrations of compound by a log-probit analysisprogram using the statistical software GraphPad Prism 4 (GraphPadSoftware, La Jolla, Calif.).

SNAP-25 Gel Cleavage Assay by Recombinant Light Chain E (rELC)

Thirteen M recombinant SNAP-25 (List Biological Laboratories, Inc.,Campbell, Calif.) was incubated with 6 μM recombinant BoNT/E LC (rELC)and 200 μM compound (dissolved in DMSO at 10× the final assayconcentration), and incubated at 37° C. for 30 min. The positive controlhad rELC and DMSO. Inhibitors (240 μM) and rELC (6.0 μM) were added torecombinant SNAP-25 (S25; 12.9 μM) and incubated at 37° C. for 30 min.Reactions were stopped by adding SDS-PAGE buffer and heating for 5 minat 70° C. Samples were run on 12% Nu-PAGE Bis Tris gels (Invitrogen,Carlsbad, Calif.) and stained by Simply Blue (Invitrogen, Carlsbad,Calif.) (see FIG. 2A).

Carboxypeptidase A (CPA) Assay

The CPA plate assay was performed according to manufacturer's (Sigma,St. Louis, Mo.) recommendation. Briefly, the mixture contained samplereaction buffer, ultrapure water, varying concentrations of compoundsdissolved in DMSO, and CPA. The positive control had CPA and DMSO, andthe negative control contained CPA+CPA inhibitor from potato tuber. Theplate was incubated for 5 minutes at 25° C. and the reaction was stoppedby the addition of stop solution. The absorption at 350 nm was read andCPA activity was calculated. Percent inhibition was determined bycomparing the CPA activity produced in the reactions with CPA alone andreactions containing CPA+test compound. Values are averages of twoindependent determinations, each in triplicate.

Cell Culture Assay

The cell culture assay was based on the procedures of Yowler et al. (49)and Boldt et al. (7). both of which are incorporated by referenceherein. Briefly, cells of the murine cholinergic neuroblastoma cell lineNeuro-2a (N2a) (ATCC # CCL-131) were incubated in Eagle's MinimumEssential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS),10 mM HEPES, 1% L-glutamine, 100 U/ml penicillin, and 100 μg/ml ofstreptomycin, in a 75-cm² cell culture flask at 37° C. in an atmosphereof 5% CO₂ and 95% air. Upon reaching 70-80% confluency, the medium wasremoved and the cells were washed with Dulbecco's phosphate-bufferedsaline (DPBS) without Ca⁺⁺ or Mg⁺. Cells were pelleted, diluted to1.5×10⁵ cells/ml and plated in 6-well cell culture plates (2 ml/well).After incubation for 48 h at 37° C. in an atmosphere of 5% CO₂ and 95%air, the medium was removed and replaced with serum-free medium, and thecells were grown for an additional 24 h.

The inhibitor (0.6-0.9 μl of the initial stock of 16.7 mM in 100% DMSO)was mixed with 1.5 μl BoNT/A (1 mg/ml) in a total volume of 2.4 μl andincubated for 30 min at 37° C. The toxin+DMSO (control) ortoxin-inhibitor mixture was added to 1 ml of EMEM without FBS to bringthe final concentration of BoNT/A toxin to 10 nM. The concentration ofBoNT/A was calibrated to produce ≥50% cleavage of the substrate SNAP-25in a 24 h incubation at 37° C. In all samples, including controls, thefinal concentration of DMSO was <0.09%.

At the end of the incubation, the medium was removed, and the cells werelysed with CelLytic™ M (Sigma, St. Louis, Mo.). Samples were run on 12%NuPAGE Bis-Tris gels (Invitrogen) and subjected to western blot analysis(see FIGS. 2B and 2C) using anti SNAP-25 monoclonal antibody (CRP,Inc.), followed by goat anti-mouse horseradish-peroxidase-conjugatedsecondary antibody (KPL, Inc.). Samples were visualized using ECLAdvance Western Blotting Detection Kit (GE Healthcare). Signals werequantitated using the UN-SCAN-IT Gel™ software (Silk Scientific, Orem,Utah). Data presented are representative of results from threeindependent assays.

Mouse Phrenic Nerve Hemidiaphragm Assay (MPNHDA)

The MPNHDA was conducted based on the procedures of Sheridan et al.(42), incorporated herein by reference. Female CD-1 mice (20-25 g) wereeuthanized with CO₂ and their diaphragms with attached phrenic nerveswere removed. The diaphragms were then divided into two hemidiaphragmswith each section complete with a phrenic nerve and myoneural junction.Each hemidiaphragm was attached to an isometric force transducer (FohrMedical Instruments, Seeheim, Germany), and its phrenic nerve wassecured to a stimulating electrode. The nerve-muscle preparations wereimmersed in separate 10-ml tissue baths containing Tyrode's buffer (1.8mM CaCl₂, 1 mM MgCl₂, 2.7 mM KCl, 137 mM NaCl, 0.4 mM NaH₂PO₄, 12 mMNaHCO₃, and 6 mM glucose), pH 7.2-7.4 (Sigma, St. Louis, Mo.). A mixtureof 95% air/5% CO₂ gas was passed through the Tyrode's buffer. The tissuebaths were kept at 37° C.

Each phrenic nerve was stimulated with single supramaximal pulses (SD9Stimulators Grass Instruments, Warwick, R.I.) through a Powerlab/4sp andBridge Amp relay (ADInstruments, Inc., Colorado Springs, Colo.) of 0.3msec duration at 0.03 Hz. The twitch tensions were digitally recorded byChart software (ADInstruments, Inc., Colorado Springs, Colo.) Maximaltwitch tensions were adjusted to 0.75-1.0 g, and the samples were runfor 20-30 min to reach a sTable 2aseline. The inhibitor (dissolved inDMSO at 2× the final assay concentration) was mixed with 60 μM BoNT/A(Metabiologics, Madison, Wis.) in 5 ml of Tyrode's buffer and incubatedfor 15-20 min at 37° C. After baseline stabilization, thetoxin-inhibitor mixture was added to a 10-ml bath with an additional 5ml of Tyrode's buffer, bringing the final concentration of BoNT/A toxinto 30 μM. The concentration of BoNT/A neurotoxin was previouslycalibrated to induce a 50% loss of twitch tension in approximately 60min. In all samples, including the controls, the final concentration ofDMSO was 0.3%.

For each experiment, four tissue baths were used. One bath was theBoNT/A toxin-only control. A second bath was an assay control withouttoxin or inhibitor. The third and fourth baths contained toxin plus twodifferent concentrations of inhibitor. Adding the toxin or thetoxin/inhibitor mixture to the bath initiated the beginning of datacollection, which continued for 5 h or until muscle twitch tensionceased.

For all preparations, neurotoxin-induced paralysis was measured as a 50%loss of twitch tension evoked by nerve stimulation. Estimates ofstatistical significance were based on unpaired, two-tailed t-test, witha P value of <0.05 considered significant, as previously reported (13,42, 43). Statistical analysis was performed using SigmaPlot 10 (SystatSoftware, San Jose, Calif.).

Procedures used to obtain mouse tissues were conducted in compliancewith the Animal Welfare Act and other U.S. federal statutes andregulations relating to animals and experiments involving animals, andadhered to principles stated in the Guide for the Care and Use ofLaboratory Animals, National Research Council, 1996. The facility wherethis research was conducted is fully accredited by the Association forAssessment and Accreditation of Laboratory Animal Care International.

Procedure for the Synthesis of 84094 Analogs

The general synthesis procedure is described in Fernando et al. (16a)and Phillips (32a), both of which are incorporated herein by reference.Briefly, these synthetic targets are accessed via the Betti reaction, aone step single pot reaction that is a modification of classic Mannichchemistry. The desired aromatic amine (1.0 mmol) is dissolved in 3 mL ofethanol in a small flask. To this, the desired aldehyde (1.0 mmol) isadded dropwise. The solution was allowed to stand for 15 to 20 minutesto promote the formation of the Mannich base. The desired quinolinol(1.0 mmol) is then dissolved in a minimal amount of ethanol with gentleheating, and the resulting solution is added to the reaction flask. Thereaction mixture is then allowed to stand until solid precipitate forms,or until thin layer chromatography indicates that no further product isbeing formed. In the case of limited solubility of the reagents ornucleophilicity of the amine component, a microwave reactor is used.Depending upon the reactants used the timeframe for the precipitation ofsolid product ranges from a few hours to several months. Purification ofthe solid precipitate or the crude reaction mixture via reverse-phasevacuum liquid chromatography using gradient elution with MeCN—H₂Omixtures yields the pure desired product in moderate to high yield.

Example 1: Identification of Small-Molecule Inhibitors of BotulinumNeurotoxins

The flow chart shown in FIG. 1 outlines the overall strategy for theidentification of small-molecule inhibitors of BoNT/A. Focus was placedon serotype A since it is the most prevalent and well-studied among thevarious serotypes in human intoxication. In silico screening was used toidentify BoNT/A inhibitors. Selected compounds were tested forinhibition of the protease activity of BoNT/A LC. Compounds that passedthe HPLC screens were advanced to in vitro and ex vivo assays.

Virtual Screening of the NCI Database

The NCI database was chosen for virtual screening on the basis of thefollowing considerations. First, it constitutes the largest freelyavailable, public domain chemical structure database with >250,000compounds (48). Second, it contains a high number of unique andstructurally diverse compounds. Thus, the NCI database provides anopportunity to discover lead compounds (48).

The compounds from the NCI database were docked into the active site inone of the protomers of BoNT/A LC (PDB code: 1E1H) (39) after removingthe peptide occupying the active site in the protomer. The top scoring500 compounds were evaluated in more detail; the list was narrowed to100 structurally-diverse compounds that interacted well with the activesite Zn and demonstrated a good fit in the BoNT/A LC binding site.

HPLC-BoNT/a LC Protease Assay

Out of 100 tested, eight compounds were selected that inhibited BoNT/A,as well as, rALC by more than 60% and 30% at 200 μM and 20 μM,respectively (Table 1). These compounds had diverse chemical structureswith molecular weights ranging from 285 to 622. To determine the actualeffectiveness of the compounds predicted by the virtual screening, anHPLC-based enzymatic assay was performed in the presence and absence ofinhibitor at 20 μM and 200 μM using full-length recombinant BoNT/A LC(rALC). The substrate peptide for BoNT/A LC protease activity consistedof residues 187-203 of SNAP-25, SEQ ID NO: 2, and BoNT/A LC catalyzesthe hydrolysis between residues Q197 and R198 (37). In this study, focuswas placed on the quinolinol lead NSC 1010. Selection of this compoundwas based on: (1) NSC 1010 was very potent against rALC; (2) NSC 1010failed to inhibit BoNT serotype B light chain (not shown), suggestingits selectivity for serotype A; (3) there are quinolinols in clinicaltrials for Alzheimer's disease and cancer (22, 33, 34); and (4)quinolinol-based drugs such as linolasept and vioform (generic name:clioquinol) are available in the market.

TABLE 1 Structures of selected hits and percent inhibition againstrecombinant full-length BoNT/A light chain (rALC)^(a).

^(a)Structures were obtained from the NCI Developmental TherapeuticsProgram website (See world wide web page atdtp.nci.nih.gov/index.html/). Compounds were tested in an HPLC-basedassay using recombinant full-length BoNT/A LC (140 nM) in the presenceof 0.8 mM 17-mer SNAP-25 peptide substrate. Percentages of inhibition at200 and 20 μM, respectively, are indicated in parentheses and weredetermined by the amounts of peptide substrate cleaved in the presenceor absence of inhibitors under the same conditions.

Screening and Testing Analog Compounds

To verify chemical identity, NSC 1010 was synthesized, and its structureand purity confirmed using standard techniques in the art. Furtherevaluation of this compound revealed that it was toxic to neuroblastomaN2a cells in cellular assays at ≥10 μM. Similarity searches wereperformed of Sigma, Chembridge and NCI databases to look for non-toxicanalogs. Fifty-five analogs were identified and synthesized; structuresof the compounds that were highly potent against the protease activityof BoNT/A (>75% inhibition) are shown in Table 2. Testing of theseanalogs in HPLC-based assays demonstrated five analogs (NSC 84094, NSC84096, CB 7967495, CB 7969312, and CB 7968218) to be more potent thanthe original hit NSC 1010 and, of equal importance, non-toxic to cells(see Table 3 below). These five analogs constitute the final compoundsthat were subsequently characterized in cell- and tissue-based assays(see Examples 2 and 3). These compounds are racemates. Whether one orboth of the enantiomers contributed to the inhibition is unknown sincethe docking poses for the two enantiomers of NSC 84096 in the activesite of BoNT/A LC were different but gave comparable scores (not shown).Table 2. Two dimensional structures of compounds displaying >75%inhibition (at 200 μM concentration) against BoNT/A light chain.Identification numbers correspond to those assigned in the NCI andChembridge databases.

TABLE 2 Two dimenstional structures of compounds displaying >75%inhibition (at 200 μM concentration) against BoNT/A light chain.Identification numbers correspond to those assigned in the NCI andChembridge databases.

NSC 84086

NSC 84094

CB 79679495

NSC 1010

NSC 84090

NSC 84096

NSC 84087

NSC 84093

NSC 84087

CB 7969312

CB 7967601

CB 7967682

CB 7968687

CB 7969312

CB 7969927

CB 7970161

CB 6372490

CB 7628245

CB 7633178

CB 6633504

CB 6637043

CB 6381661

CB 7925368

CB 6378057

CB 6380823

CB 6636098

NSC 84093

CB 7968218

To compare the relative inhibitory potencies of the five analogs,HPLC-based assays were performed using two recombinant forms of BoNT/Alight chain: full-length (rALC) and truncated (1-425; tALC). Under assayconditions, rALC was at least 4× more active than tALC. The five analogswere very effective against both forms of BoNT/A LC, demonstrating IC₅₀values ranging from 1.6-4.7 μM and 1.5-5.0 μM for rALC and tALC,respectively (Table 3).

TABLE 3 Structural formula and IC₅₀ values of selected analogs againstrecombinant full-length BoNT/A light chain (rALC) and truncated (1-425)A ligh chain (tALC)^(a)

^(a)Structures were obtained from the NCI Developmental TherapeuticsProgram website (see the world wide web page fordtp.nci.nih.gov/index.html/), and those of CB compounds were fromChembridge screening compounds and building blocks (see the world wideweb page for hit2lead.com/). All compounds tested were racemates. Thestructure and purity of these analogs were confirmed by LC-MS and NMR.The IC₅₀ values for rALC and tALC are indicated in parenthesis,respectively, and were determined from nine concentrations of eachinhibitor using GraphPad Prism 4 (GraphPad Software, La Jolla, CA). rALC(140 nM final concentration) or tALC (620 nM) was incubated with 0.8 mM17-mer SNAP-25 peptide substrate and varying concentrations of inhibitor(dissolved in DMSO at 10X final concentration) at 37° C. for 5 min in 50mM HEPES, pH 7.3. Reactions were stopeed by adding 0.7% TFA and analyzedby reverse-phase HPLC.

Having established that these five analogs were very potent againstBoNT/A LC, their effects were tested on two related BoNT endopeptidases:recombinant full-length BoNT/B light chain (rBLC) and BoNT/E light chain(rELC). The five compounds showed cross-reactivity against rBLC, withboth NSC 84096 and NSC 84094 exhibiting the least inhibition at 6.9% and8.8%, respectively (Table 4). However, none of the compounds (at 240 mM)inhibited rELC in gel cleavage assays (FIG. 2A). Although additionalBoNT light chain serotypes would need to be tested before concludingthat these five compounds are more selective for BoNT/A, thispossibility deserves consideration.

TABLE 4 Percent inhibition of selected small-molecules againstrecombinant BoNT/A light chain (rALC) and BoNT/B light chain (rBLC) ^(a)% Inhibition Against Compound rALC rBLC BoNT/A BoNT/B NSC 84096 91.9 ±1.7  6.9 ± 2.0 77.8 ± 2.0 55.0 ± 0.1 NSC 84094 97.4 ± 2.2  8.8 ± 0.885.1 ± 0.4 77.1 ± 0.0 CB 7967495 92.7 ± 1.5 30.2 ± 2.4 90.6 ± 2.2 86.8 ±0.0 CB 7968218 92.8 ± 0.8 16.8 ± 2.2 95.6 ± 2.1 90.2 ± 0.0 CB 796931296.2 ± 1.2 31.2 ± 3.0 71.0 ± 0.7 85.0 ± 0.0 ^(a) HPLC-based proteaseassays were conducted at 37° C. using various inhibitors at 20 μM finalconcentration. rALC assays contained 50 mM HEPES (pH 7.3), 0.8 mMSNAP-25 peptide substrate, test compound in DMSO, and rALC (110-140 nM).The assay for rBLC contained 50 mM HEPES (pH 7.3), 1 mM DTT, 0.4 mM35-mer VAMP peptide substrate, test compound in DMSO, and rBLC (30-40nM). Inhibitors were diluted into the reaction mixture containing thesubstrate, followed by the addition of LC (i.e., inhibitor and LC werenot pre-incubated). Reactions were stopped by acidification by TFA, andanalyzed by reverse-phase HPLC. Data represent mean ± SD from 2independent assays.

Further experimental studies revealed that all five compounds (atconcentrations up 20 μM) showed no inhibitory effect on the zincprotease carboxypeptidase (CPA), while the control inhibitor from potatotuber exhibited 100% inhibition at concentrations equal to or greaterthan 0.187 μM (not shown). This finding precludes the possibility thatthe inhibition of BoNT/A endopeptidase by these analogs was due tononspecific chelation.

Further synthesis and testing of novel analogs of NSC 1010 provided newchemical entities with comparable or improved potency (Table 5).

TABLE 5 Previously unreported analogs with potent activity againstBoNT/A % Inhibition against BoNT/A IC₅₀ Compound Chemical Structure (5μM) (μM) 5116-047A

83.0 1.4 5116-048A

87.0 0.8 5116-050A

86.7 1.2 5116-051A

91.0 <1.0 5116-052A

97.5 <1.0 4874-051A

94.0 2.2

Example 2: Inhibition in Cell-Based Assay

To this point, it has been demonstrated that five compounds inhibit theenzymatic activity of BoNT/A in vitro. To examine the likelihood thatthese compounds might be useful antibotulinum therapeutics, studies wereperformed in other systems. One of these used murine neuroblastoma N2acells to evaluate the ability of these compounds to protectBoNT/A-mediated cleavage of intracellular SNAP-25. BoNT/A binds to N2acells and the translocated LC cleaves SNAP-25 in these cells. The extentof cleavage of SNAP-25 in N2a cells by BoNT/A was determined by westernblot analysis using monoclonal antibodies against SNAP-25. As shown inFIG. 2B, all five lead analogs at 15 μM, exhibited complete protectionof BoNT/A-mediated SNAP-25. At 10 μM, three of these compounds (CB7967495, NSC 84094, CB 7969312) showed near-to-complete inhibition ofSNAP-25 cleavage while the other two (NSC 84096 and CB 7968218) afforded66% and 68% protection, respectively (FIG. 2C).

Example 3: Activity in Mouse Phrenic Nerve Hemidiaphragm Assay

Encouraged by these findings, the efficacy of these analogs was examinedat the tissue level using mouse phrenic nerve hemidiaphragm preparationswhose intact neuromuscular junction permits the monitoring of theeffectiveness of an inhibitor by recording muscle twitch tension. In theex-vivo mouse phrenic nerve hemidiaphragm assay (MPNHDA), the time toonset of neuromuscular block is a concentration-sensitive event, withblockade occurring earlier with higher toxin concentrations (13, 40,41). Changes in the onset of muscle paralysis with a fixed concentrationof toxin are used to indicate the activity of candidate BoNTtherapeutics.

Results from MPNHDA experiments demonstrated that applying effectiveconcentrations of compounds significantly delayed (P<0.01) the onset oftoxin-induced paralysis (Table 6). While neuromuscular preparationsexposed to BoNT/A alone (control) produced an average paralytic time of65.7±7.80 min, those that were exposed to inhibitors at effectiveconcentrations ranged from 216.3-281.0 min. The effective concentrationsthat caused a delay in time to 50% loss of twitch tension for the fiveinhibitors ranged from 0.5 μM-20 μM. Moreover, the muscle twitch-tensiontime courses of the five inhibitors at their effective concentrationsduring the entire assay period (300 min) were comparable to the NoToxin-control group (FIG. 3). By comparison, the peptide BoNT/Ainhibitor Ac-CRATKML-NH₂ (SEQ ID NO: 1) (K_(i)=1.9 μM) (38) did notcause a delay in paralytic time at 20 μM, and showed muscletwitch-tension time courses that were similar to that of BoNT/A alone(control) (FIG. 3). This is the first report of BoNT/A small-moleculeinhibitors that showed activity in an ex vivo assay.

TABLE 6 Effect of small-molecule inhibitors in protecting BoNT/A-inducedneuromuscular block in mouse phrenic nerve hemidiaphragm assays. AverageTime Concentration to 50% Loss of Inhibitor (μM) Twitch Tension(min)^(a) Without BoNT/A — >300.00 With BoNT/A — 65.70 ± 7.80  BoNT/A +NSC 84096 20.0 216.3 ± 20.2^(b) 0.10 59.7 ± 4.9^(d) BoNT/A + NSC# 8409410.0 266.3 ± 19.6^(b) 0.25 72.0 ± 5.5^(c) BoNT/A + CB 7967495 5.00 216.3± 25.2^(b) 1.00 58.7 ± 7.5^(c) BoNT/A + CB 7968218 10.0 281.0 ± 19.0^(b)0.10  66.3 ± 10.6^(c) BoNT/A + CB 7969312 0.50 271.0 ± 29.0^(b) 0.10 91.7 ± 19.6^(c) BoNT/A + SEQ ID NO: 1 20.0 52.70 ± 9.30^(c) 5.00  65.0± 11.5^(c) ^(a)Specified concentrations of small-molecule inhibitors andBoNT/A were added to hemidiaphragm preparations, and isometriccontractions of the electrically-stimulated muscles were recorded andanalyzed. The time required to 50% of loss of twitch tension (paralytichalf-time) was determined. Controls (with and without BoNT/A) used thesame amount of DMSO (0.3% final concentration) as those with inhibitors.Data are means ± SE (n = 3 for small-molecules and Ac-CRATKML-NH₂ (SEQID NO: 1); n = 18 for With BoNT/A and without BoNT/A control groups).^(b)P < 0.01, highly significant; ^(c)P > 0.05, not significant fromthose recorded with BoNT/A control. Statistical analysis was performedusing SigmaPlot 10 (Systat Software, San Jose, CA). ^(d)P > 0.05, notsignificant from those recorded with BoNT/A alone. Statistical analhysiswas performed using SigmaPlot 10.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by thefollowing claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A composition having Formula 1:

wherein R₁, R₂, and R₃, being the same or different, are each selectedfrom the group consisting of a H, a C₁ to C6 straight or branched alkylgroup, an aryl group, a halogen, NR₅R₆, NO₂, CH₂ CH₂OH, CHO, COOH, CN,SO₃H, and SO₂NR₇R₈; wherein R₅, R₆, R₇ and R₈ being the same ordifferent, are each selected from the group consisting of a H, a C₁ toC₆ straight or branched alkyl group, and an aryl group; wherein R₄ isselected from the group consisting of a straight or branched alkyl groupand an aryl group; wherein Ar₁ is selected from the group consisting of5- or 6-membered aromatic rings, 5- or 6-membered heteroaromatic rings,polycyclic aromatic ring systems, and heteroaromatic ring systems;wherein X is selected from the group consisting of a O, OCH₂, S, SCH₂,NR₉, NR₉CH₂, NR₉CO, and CH₂; and wherein R₉ is selected from the groupconsisting of a H, a C₁ to C₆ straight or branched alkyl group, and anaryl group.
 2. The composition of claim 1 wherein the halogen isselected from the group consisting of F, Cl, Br, and I.
 3. Thecomposition of claim 1 wherein the aryl group is a phenyl, biphenyl,thiophenyl, or indolyl ring.
 4. The composition of claim 1 furthercomprising a pharmaceutically acceptable carrier, excipient, diluent, oradjuvant.
 5. The composition of claim 1, which further includes apharmaceutically acceptable salt of the compound of Formula
 1. 6. Amethod of inhibiting a botulinum neurotoxin in a subject comprisingadministering to the subject a composition comprising at least onecompound of Formula 1

wherein R₁, R₂, and R₃, being the same or different, are each selectedfrom the group consisting of a H, a C₁ to C₆ straight or branched alkylgroup, an aryl group, a halogen, NR₅R₆, NO₂, CH₂OH, CHO, COOH, CN, SO₃H,and SO₂NR₇R₈; wherein R₅, R₆, R₇, and R₈, being the same or different,are each selected from the group consisting of a H, a C₁ to C₆ straightor branched alkyl group, and an aryl group; wherein R₄ is selected fromthe group consisting of a straight or branched alkyl group and an arylgroup; wherein Ar₁ is selected from the group consisting of 5- or6-membered aromatic rings, 5- or 6-membered heteroaromatic rings,polycyclic aromatic ring systems, and heteroaromatic ring systems;wherein X is selected from the group consisting of a O, OCH₂, S, SCH₂,NR₉, NR₉CH₂, NR₉CO, and CH₂; and wherein R₉ is selected from the groupconsisting of a H, a C₁ to C₆ straight or branched alkyl group, and anaryl group.
 7. The method of claim 6 wherein the composition includestwo or more compounds of Formula
 1. 8. The method of claim 6 wherein thecomposition includes a pharmaceutically acceptable salt of a compound ofFormula
 1. 9. The method of claim 6 wherein the composition furthercomprises a pharmaceutically acceptable carrier, excipient, diluent, oradjuvant.
 10. The method of claim 6 wherein the botulinum neurotoxin isa Clostridium botulinum serotype A neurotoxin, and inhibiting thebotulinum neurotoxin inhibits or prevents a Clostridium botulinuminfection.
 11. The method of claim 6 wherein the subject is an animal.12. The method of claim 6 wherein the subject is a human.
 13. A kit fortreating a subject exposed to a botulinum neurotoxin comprising acomposition with at least one compound of Formula 1 in a container, andinstruction on administering the at least one compound, wherein Formula1 comprises

wherein R₁, R₂, and R₃, being the same or different, are each selectedfrom the group consisting of a H, a C₁ to C6 straight or branched alkylgroup, an aryl group, a halogen, NR₅R₆, NO₂, CH₂ CH₂OH, CHO, COOH, CN,SO₃H, and SO₂NR₇R₈; wherein R₅, R₆, R₇ and R₈ being the same ordifferent, are each selected from the group consisting of a H, a C₁ toC₆ straight or branched alkyl group, and an aryl group; wherein R₄ isselected from the group consisting of a straight or branched alkyl groupand an aryl group; wherein Ar₁ is selected from the group consisting of5- or 6-membered aromatic rings, 5- or 6-membered heteroaromatic rings,polycyclic aromatic ring systems, and heteroaromatic ring systems;wherein X is selected from the group consisting of a O, OCH₂, S, SCH₂,NR₉, NR₉CH₂, NR₉CO, and CH₂; and wherein R₉ is selected from the groupconsisting of a H, a C₁ to C₆ straight or branched alkyl group, and anaryl group.
 14. A composition having Formula 2

wherein X is selected from the group consisting of a O, S, Se, and NH;wherein Y is selected from the group consisting of a CH or N; whereinAr₁ is selected from the group consisting of a monocyclic or bicyclicaromatic or heteroaromatic ring system, a biphenyl or bipyridyl ringsystem, a bridged biphenyl or bipyridyl ring system, wherein any ringsystem may be further substituted by one or more substitution selectedfrom the group consisting of a halogen, NR₁R₂, OR₃, SO₂NR₄R₅, SR₆, andR₇; and wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇, being the same ordifferent, are each selected from the group consisting of H, a C₁ to C₆straight or branched chain or cycloalkyl ring, a C₁ to C₆ straight orbranched chain acyl group, and an aryl group.
 15. A method of inhibitinga botulinum neurotoxin in a subject comprising administering to thesubject a composition comprising at least one compound of Formula 2

wherein X is selected from the group consisting of a O, S, Se, and NH;wherein Y is selected from the group consisting of a CH or N; whereinAr₁ is selected from the group consisting of a monocyclic or bicyclicaromatic or heteroaromatic ring system, a biphenyl or bipyridyl ringsystem, a bridged biphenyl or bipyridyl ring system, wherein any ringsystem may be further substituted by one or more substitution selectedfrom the group consisting of a halogen, NR₁R₂, OR₃, SO₂NR₄R₅, SR₆, andR₇; and wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇, being the same ordifferent, are each selected from the group consisting of H, a C₁ to C₆straight or branched chain or cycloalkyl ring, a C₁ to C₆ straight orbranched chain acyl group, and an aryl group.
 16. The method of claim 15wherein the botulinum neurotoxin is a Clostridium botulinum serotype Aneurotoxin, and inhibiting the botulinum neurotoxin inhibits or preventsa Clostridium botulinum infection.
 17. The method of claim 15, whereinthe composition includes two or more compounds of Formula
 2. 18. Themethod of claim 15, wherein the composition includes a pharmaceuticallyacceptable salt of a compound of Formula
 2. 19. The method of claim 15,wherein the composition further comprises a pharmaceutically acceptablecarrier, excipient, diluent, or adjuvant.
 20. A kit for treating asubject exposed to a botulinum neurotoxin comprising a composition withat least one compound of Formula 2 in a container, and instruction onadministering the at least one compound, wherein Formula 2 comprises

wherein X is selected from the group consisting of a O, S, Se, and NH;wherein Y is selected from the group consisting of a CH or N; whereinAr₁ is selected from the group consisting of a monocyclic or bicyclicaromatic or heteroaromatic ring system, a biphenyl or bipyridyl ringsystem, a bridged biphenyl or bipyridyl ring system, wherein any ringsystem may be further substituted by one or more substitution selectedfrom the group consisting of a halogen, NR₁R₂, OR₃, SO₂NR₄R₅, SR₆, andR₇; and wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇, being the same ordifferent, are each selected from the group consisting of H, a C₁ to C₆straight or branched chain or cycloalkyl ring, a C₁ to C₆ straight orbranched chain acyl group, and an aryl group.